It is the long-range goal of this work to clarify the nature of cellular molecules responsible for the malignant phenotype of chemically transformed cells by using new techniques in molecular biology. When the protein molecules were compared by two-dimensional gel electrophoresis between the chemically transformed human fibroblasts and normal parent cell, one new polypeptide was recognized in the transformed cells. This novel polypeptide was identified as a product of mutated B-actin gene by immunoprecipitation with anti-actin antibody, comparison of its tryptic peptide patterns, isolation of mRNA coding for the new protein, cloning of DNA complementary to actin mRNA from the transformed cells, determination of size of actin mRNAs, complete amino acid sequencing of normal and altered B-actin molecules, and Southern blotting analysis of actin gene in the transformed cells using a recombinant DNA containing actin cDNA. These results provide the first molecular evidence for an occurrence of mutation in the chemically transformed cells. Some results were obtained which indicate the close correlation of production of mutated B-actin with the expression of the transformed phenotypes.